1. Field of the Invention
The present invention concerns the use of a modified form of fibrinogen as a fibrin-like substance in immunological and biochemical assays and procedures that require soluble fibrin monomer, soluble fibrin or fibrin.
2. Background Information
The soluble plasma protein fibrinogen is the precursor from which insoluble fibrin networks of blood clots form. This process is well known. Fibrinogen is activated by thrombin and the activated fibrinogen polymerizes to form the clot. Activation occurs through cleavage of the fibrinogen A.alpha.15Arg-16Gly bond, which releases the amino-terminal fibrinopeptide A, and the B.beta.14Arg-15Gly bond, which releases the amino-terminal fibrinopeptide B. It is believed that the new amino-termini act as polymerization domains that interact with complementary polymerization sites at the carboxy-terminal region of neighboring molecules, thus enabling activated fibrin monomers to associate by aligning in a double-stranded protofibril. Protofibrils grow in length and associate laterally to form thicker fibrin fibers. These fibers also associate to form thicker fibrin strands of a clot network.
The formation of fibrin in vivo influences many physiological processes related to hemostasis, such as the activation of the clot stabilizing enzyme (factor XIII), the control of fibrinolysis and endothelial cell secretion. Laboratory studies of these processes typically involve analysis of the phenomenon in the presence of fibrin. The stimulatory (or required) effect of fibrin on the activity is documented. Since fibrin polymerizes, a process which, in neutral buffers, is accompanied by formation of a precipitate or aggregate, the use of fibrin in biological and clinical assays has heretofore been limited. A solubilized form of fibrin, or fragments of fibrin formed by proteolytic or chemical degradation of fibrin have been used in most studies, since the clotted form of fibrin is difficult to handle and cannot be easily quantitated.
Solubilized fibrin is routinely prepared from clotted fibrin by dissolving the clot in buffers containing high concentrations of chaotropic denaturing reagents, e.g., urea, guanidine hydrochloride, sodium bromide or acid (e.g., acetic acid). Only fibrin that has not been stabilized by factor XIII can be dissolved in buffers containing any of these agents. Fibrin solubilized by such methods remain in solution only in the presence of the acid or chaotrope containing buffers. These conditions destroy the biological properties of most other proteins, enzymes and cells of interest, and for this reason acid or chaotrope-treated soluble fibrin has only limited applications.
Soluble fibrin is also prepared by converting fibrinogen to fibrin in the presence of polymerization inhibitors, e.g., the peptide Gly-Pro-Arg-Pro (Laudano, A. P., Cottrell, B. A. and Doolittle, R. F., (1983) Synthetic Peptides Modeled on Fibrin Polymerization Sites, N.Y. Acad. Sci., Vol. 408, pp. 315-329 and reference cited within) or fibrinogen fragment D. In this case, the activation of fibrinogen usually is carried out only on the A.alpha.-chain using snake venom enzymes, e.g., batroxobin (Wiman, B. and Ranby, M., (1986), Determination of Soluble Fibrin in Plasma By a Rapid and Quantitative Spectrophotometric Assay, Thromb. and Haemostas, 55, 189-193). Only one set of polymerization sites is activated by this method and a single polymerization inhibitor can be used to effectively prevent the aggregation of the fibrin, e.g., DESAFIB.TM., from American Diagnostica, Inc., Greenwich, Conn. The fibrin remains soluble in physiological buffers, however, only if there is a suitable high concentration of the polymerization inhibitor present in the solution. Fibrin precipitation occurs if the concentration of the polymerization inhibitor is lowered by dilution.
Because of the difficulty of solubilizing fibrin under physiological conditions, any new form of fibrin, or a substance with the properties of fibrin, that does not require special conditions for maintaining solubility would be very useful, especially for use in biochemical and immunological assays that require fibrin monomer or soluble fibrin.
Procyk and Blomback have described the preparation of modified forms of fibrinogen by disulfide bond reduction which have a prolonged clotting time when treated with thrombin (Procyk, R. & Blomback, B. (1990) Disulfide Bond Reduction in Fibrinogen: Calcium Protection and Effect on Clottability. Biochemistry 29, 1501-1507). The modified fibrinogen was prepared by mild reduction in the absence of calcium ions and formed a gel only by a mechanism involving oligomerization and crosslinking catalyzed by factor XIII. Until now, however, nothing was known about the properties of this material, its stability, solubility, or suitability in applications requiring soluble fibrin or soluble fibrin monomers.
Applicants have discovered that the modified fibrinogen has substantial biochemical and immunological equivalency to fibrin and use this discovery to invent useful applications of the modified fibrinogen in assays and procedures. This invention has special applications in diagnostic determinations of coagulation and/or fibrinolysis which require a form of soluble fibrin or fibrinogen fragments for the assay procedure.